Summary: Treatment of neonatal anemia is largely based on measured hemoglobin concentrations (Hb). Historically, neonatologists developed a conservative approach to blood management, using lower Hb thresholds to trigger transfusions. However, our recent multicenter prospective cohort investigation demonstrated that significant anemia in preterm infants (Hb ?8g/dL) is associated with the development of necrotizing enterocolitis (NEC), a serious intestinal disease and major cause of death in preterm neonates. Our long-term objective is to identify key mechanisms that regulate anemia-induced alterations in neonatal immunity that contribute to gut inflammation and injury and thus may predispose neonates to inflammatory conditions such as NEC. Our central hypothesis is that variability in anemia-induced alterations in immunosuppressive erythroid progenitors (IEPs) and hypoxia-induced inflammation can differentially impact immune function in the gut, directly predisposing neonates to gut injury that may cause NEC. Our hypothesis is formulated on the basis of our recent discovery that severe anemia in preterm infants can result in impaired gut oxygenation, as measured by near infrared spectroscopy (NIRS), and significantly increased serum levels of pro-inflammatory interferon gamma (IFNg). Using a preclinical model, our data also demonstrate that anemia drives IFNg production by intestinal macrophages that induces intestinal injury, consistent with previous studies that demonstrate that IFNg can directly compromise epithelial barrier function. Importantly, anemia also induces the development of erythroid progenitors, which not only possess the ability to facilitate increased red blood cell (RBC) production, but also appear to be intrinsically immunosuppressive. Consistent with this, IEPs isolated from cord blood possess the ability to suppress macrophage activation, while removal of IEPs in our pre-clinical model exacerbates anemia- induced gut macrophage activation and intestinal injury. Taken together, these results suggest that individual variation in the hypoxic response to lower Hb values, coupled with alterations in anemia-induced IEP numbers and function, creates imbalances that alter local macrophage activity leading to distinct responses in the gut that predispose neonates to intestinal inflammation and place them at higher risk of NEC. To test our central hypothesis, we will pursue the following specific aims: Aim 1: Define the correlation between anemia and its treatment on IEP number and function, and how these relate to serum cytokines, pro-inflammatory monocyte differentiation, and markers of intestinal oxygenation, inflammation, and injury. Aim 2: Define the impact of anemia-induced IEPs on macrophage pro-inflammatory cytokine secretion, intestinal inflammation and injury following different thresholds, durations and treatments of anemia in a pre-clinical model. We think these aims provide a unique opportunity to define key factors that regulate anemia-induced alterations in intestinal inflammation and injury. In doing so, these data possess the capacity to provide important insight into the global immune impact of anemia on neonatal intestinal injury that may contribute to NEC.